N6 -substituted 9-methyladenines: a new class of adenosine receptor antagonists

ABSTRACT

Novel compounds and a method of using them to antagonize adenosine receptors are provided wherein the compounds are selected from the group of racemic mixtures or optically active compounds represented by the general formula: ##STR1## wherein R 2  is selected from the group consisting of cycloalkyl radicals having from 3 to 8, preferably 3 to 7, ring carbon atoms, alkyl radicals having from 1 to 10, carbon atoms, aralkyl radicals having from 7 to 14, preferably 7 to 10, carbon atoms, and heteroatom- and halogen-substituted derivatives thereof wherein said heteroatom may be selected from the group consisting of nitrogen, phosphorus, sulfur and oxygen; R 1  may be hydrogen or R 2 , and R 3  is selected from the group consisting of hydrogen, halogen, amine, carboxy, thio, sufonate, sulfonamide, sulfone, sulfoxamide, phenyl, alkyl-substituted amine, cycloalkyl-substituted amine, alkyl radicals having from 1 to 10 carbon atoms, and cycloalkyl radicals having from 3 to 8, preferably 5 to 6, ring carbon atoms. R 4  is selected from the group consisting of benzyl, phenyl, and alkyl groups comprising from 1 to 4 carbon atoms, wherein said alkyl group can be substituted with oxygen, for example ethers and alcohols. R 5  is selected from the group consisting of hydrogen; hydroxy; sulfonate; halogen; alkoxy and cycloalkoxy groups comprising 1 to 6 carbon atoms, wherein said alkoxy and cycloalkoxy groups can be substituted with phenyl; and amine, wherein said amine can be substituted with alkyl, cycloalkyl, or phenyl. Optically active compounds are especially active as antagonists of A 1  adenosine receptors.

This application is a continuation of application Ser. No. 08/073,916, filed Jun. 8, 1993, now abandoned, which is a continuation of application Ser. No. 07/320,708, filed Mar. 8, 1989, now abandoned, which is a continuation-in-part of application Ser. No. 07/304,346, filed Jan. 31, 1981, now abandoned, which is a continuation-in-part of application Ser No. 07/042,383, filed Apr. 24, 1987, now abandoned, the contents of each of which are fully incorporated by reference.

SUMMARY OF THE INVENTION

Novel compounds and a method of using them to antagonize adenosine receptors are provided wherein the compounds are selected from the group of racemic mixtures or optically active compounds represented by the formula: ##STR2## wherein R₂ is selected from the group consisting of cycloalkyl radicals having from 3 to 8, preferably 3 to 7, ring carbon atoms, alkyl radicals having from 1 to 10 carbon atoms, aryl radicals having from 6 to 13, preferably 6 to 10, carbon atoms, aralkyl radicals having from 7 to 14, preferably 7 to 10, carbon atoms,: and heteroatom- and halogen-substituted derivatives thereof wherein said heteroatom may be selected from the group consisting of nitrogen, phosphorus, sulfur and oxygen; R₁ may be hydrogen or R₂, and R₃ is selected from the group consisting of hydrogen, halogen, amine, carboxy, thio, sufonate, sulfonamide, sulfone, sulfoxamide, phenyl, alkyl-substituted amine, cycloalkyl-substituted amine, alkyl radicals having from 1 to 10 carbon atoms, and cycloalkyl radicals having from 3 to 8, preferably 5 to 6, ring carbon atoms. R₄ is selected from the group consisting of benzyl, phenyl, and alkyl groups comprising from 1 to 4 carbon atoms, wherein said alkyl group can be substituted with oxygen, for example ethers and alcohols. R₅ is selected from the group consisting of hydrogen; hydroxy; sulfonate; halogen; alkoxy and cycloalkoxy groups comprising 1 to 6 carbon atoms, wherein said alkoxy and cycloalkoxy groups can be substituted with phenyl; and amine, wherein said amine can be substituted with alkyl, cycloalkyl, or phenyl.

BACKGROUND OF THE INVENTION

Adenosine receptors have been divided into two subtypes, based on adenylate cyclase activity: A₁ (R_(i)) receptors mediate inhibition and A₂ (R_(a)) receptors mediate stimulation of adenylate cyclase activity. Some N⁶ -substituted adenosine analogs, like N⁶ -R-phenyl isopropyl adenosine (R-PIA) have very high affinity for A₁ adenosine receptors, but at A₂ receptors 5'-N-ethylcarboxamido-adenosine (NECA) is more potent than N⁶ -substituted analogs. Alkylxanthines, such as caffeine and theophylline, are the best known antagonists at adenosine receptors.

Adenine was generally believed to have no effect on adenosine receptor-controlled systems. However, it was found that at low concentrations adenine displays specific competitive antagonism of adenosine-induced cyclic Amp accumulation in a human fibroblast cell line. Methylation of adenine at the 9-position increases potency about 4-fold in this assay. At higher concentration, both compounds show nonspecific inhibitory activity.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of this invention are selected from the group of compounds, including the racemic mixtures or optically active compounds represented by the general formula: ##STR3## wherein R₂ is selected from the group consisting of cycloalkyl radicals having from 3 to 8, preferably 3 to 7, ring carbon atoms, alkyl radicals having from 1 to 10 carbon atoms, aryl radicals having from 6 to 13, preferably 6 to 10, carbon atoms, aralkyl radicals having from 7 to 14, preferably 7 to 10, carbon atoms, and heteroatom- and halogen-substituted derivatives thereof wherein said heteroatom may be selected from the group consisting of nitrogen, phosphorus, sulfur and oxygen; R₁ may be hydrogen or R₂, and R₃ is selected from the group consisting of hydrogen, halogen, amine, carboxy, alkyl radicals having 1 to 10 carbon atoms, cycloalkyl radicals having from 3 to 8, preferably 5 to 6, ring carbon atoms, thio, sulfonate, sulfonamide, sulfone, sulfoxamide, phenyl, alkyl-substituted amine, and cycloalkyl substituted amine. R₄ is selected from the group consisting of benzyl, phenyl, and alkyl groups comprising from 1 to 4 carbon atoms, wherein said alkyl group can be substituted with oxygen, for instance ethers and alcohols. R₅ is selected from the group consisting of hydrogen; hydroxy; sulfonate; halogen; alkoxy and cycloalkoxy groups comprising 1 to 6 carbon atoms, wherein said alkoxy and cycloalkoxy groups can be substituted with phenyl; and amine, wherein said amine can be substituted with phenyl and alkyl and cycloalkyl groups comprising 1 to 6 carbon atoms.

Preferred compounds are those wherein R₁ is hydrogen; wherein R₂ is endo-2-Norbornyl or cyclopentyl; wherein R₃ is bromine, chlorine, amino, hydrogen, thio, cyclopentyl or cyclopentylamine; wherein R₄ is methyl, ethyl, 2-hydroxyethyl, phenyl, or 2-hydroxyethoxy methyl; and wherein R₅ is hydrogen, hydroxy, chlorine or fluorine.

The following is a list of compounds, including the racemic mixtures or optically active species of which are useful in the practice of the present invention. This list is intended to be illustrative and the scope of the invention is not limited to compounds named therein:

N⁶ -Cyclobutyl-9-Methyl Adenine (MA)

N⁶ -Cyclopentyl-9-MA

N⁶ -Methylcyclopentyl-9-MA

N⁶ -Cyclohexyl-9-MA

N⁶ -Methyl-9-MA

N⁶ -3-pentyl-9-MA

N⁶ -Phenyl-9-MA

N⁶ -2-Fluorophenyl -9-MA

N⁶ -Benzyl-9-MA

N⁶ -2-Phenethyl-9-MA

N⁶ -2- (3,4,5-Trimethoxyphenyl) ethyl-9-MA

N⁶ -2-(3-Pyridylethyl)-9-MA

N⁶ -2-(3-Thienylethyl)-9-MA

N⁶ -R-1-Phenyl-2-propyl-9-MA

N⁶ -S-1-Phenyl-2-propyl-9-MA

N⁶ -(endo-2-Norbornyl)-9-MA

N⁶ -[endo-(1R,2S,4S)-2-Norbornyl]-9-MA

N⁶ -[endo-(1S,2R,4R)-2-Norbornyl]-9-MA

N⁶ -1-(2-Thienyl)-2-butyl-9-MA

N⁶ -(exo-2-Norbornyl)-9-MA

N⁶ -2,2-diphenylethyl-9-MA

N⁶ -2-phenylethyl-9-MA

N⁶ -2-(2-chlorophenyl)ethyl-9-MA

N⁶ -indanyl-9-MA

N⁶ -2-aminoethyl-9-MA

N⁶ -(N,N-Dimethylaminoethyl)-9-MA

N⁶ -R-1-phenyl-1-ethyl-9-MA

N⁶ -S-1-phenyl-1-ethyl-9-MA

N⁶ -2-thienyl-9-MA

N⁶ -(4-chloro-2-methyl phenyl)-9-MA

N⁶ -2-(3-ethylindole)-9-MA

N⁶ -(1-methyl-2-phenylethyl)-9-MA

N⁶ -(1-methyl-2-phenoxyethyl)-9-MA

N⁶ -1-carboxy-1-butyl-9-MA

N⁶ -(endo-2-norbornyl)-2-chloro-9-MA

N⁶ -(endo-2-norbornyl)-8-cyclopentyl-9-MA

N⁶ -(endo-2-norbornyl)-8-hydroxy-9-MA

N⁶ -(endo-2-norbornyl)-8-bromo-9-MA

N⁶ -[endo-(1R,2S,4S)-2-Norbornyl]-8-bromo-9-MA

N⁶ -[endo-(1S,2R,4R)-2-Norbornyl]-8-bromo-9-MA

N⁶ -(endo-2-norbornyl)-8-amino-9-MA

N⁶ -(endo-2-norbornyl)-8-carboxy-9-MA

N⁶ -cyclopentyl-8-cyclopentyl-9-MA

N⁶ -(endo-2-norbornyl)-9-[(2-hydroxyethoxy)methyl]adenine

N⁶ -(endo-2-norbornyl)-8-thio-9-MA

N⁶ -(endo-2-norbornyl)-8-chloro-9-MA

N⁶ -[endo-(1R,2S,4S)-2-Norbornyl]-8-chloro-9-MA

N⁶ -[endo-(1S,2R,4R)-2-Norbornyl]-8-chloro-9-MA

N⁶ -(endo-2-norbornyl)-8-sulfonate-9-MA sodium salt

N⁶ -(endo-2-norbornyl)-2-hydroxy-9-MA

N⁶ -(endo-2-norbornyl)-8-cyclopentylamine-9-MA

N⁶ -(endo-2-norbornyl)-8-propylamine-9-MA

N⁶ -(endo-2-norbornyl)-9-phenyl adenine

N⁶ -cyclopentyl-2-chloro-9-MA

N⁶ -phenyl-2-chloro-9-MA

N⁶ -cyclopentyl-9-phenyl adenine

N⁶ -R-1-phenyl-2-propyl-9-phenyl adenine

N⁶ -S-1-phenyl-2-propyl-9-phenyl adenine

N⁶ -[(3-chloro-endo-2-norbornyl)]-9-MA

N⁶ -phenyl-9-phenyl adenine

2-ethoxy-9-MA

2-propoxy-9-MA

2-butoxy-9-MA

2-isopropoxy-9-MA

2-(2-butoxy)-9-MA

2-(2-methyl propoxy) 9-MA

2-pentoxy-9-MA

2-(2-phenylethoxy) 9-MA

2-phenylamino-9-MA

9-hydroxyethyladenine

N⁶ -cyclopentyl-9-benzyl adenine

N⁶ -cyclohexyl-9-ethyl adenine

It is essential that the compound selected for use in the method of the present invention be an optically active compound or racemic mixture thereof capable of inhibiting activity of adenosine receptors, e.g., in a human. In particular it is found that the isomers of N⁶ -(endo-2-Norbornyl)-9 methyladenines are especially preferred for inhibiting activity of A₁ adenosine receptors. One group of these preferred stereoisomers has the general formula ##STR4## wherein X is halogen or hydrogen. This group of preferred stereoisomers is designated as the N⁶ -[endo(1R,2S,4S)-2-Norbornyl]-9 methyladenines. For example, N⁶ -[endo(1R,2S,4S)-2-Norbornyl]-8-bromo-9-MA and N⁶ -[endo(1R,2S,4S)-2-Norbornyl]-8-chloro-9-MA. The enantiomers of these compounds are the stereoisomers, having the general formula ##STR5## wherein X is hydrogen or halogen. The latter group of preferred stereoisomers is designated as the N6[endo-(1S,2R,4R)-2-Norbornyl]-9-methyladenines, for example N⁶ [endo-(1S,2R,4R)-2-Norbornyl]-8-bromo-9-methyladenine and N⁶ [endo-(1S,2R,4R)-2-Norbornyl]-8-chloro-9-methyladenine.

The preparation of 9-methyl adenines is well known. See R. K. Robins, K. J. Dille and B. E. Christensen, J. Org. Chem., 19, 930 (1954); R. K. Robins and H. H. Lin, J. Am. Chem. Soc., 79, 490 (1957; and J. A. Montgomery and Carroll Temple, Jr., J. Am. Chem. Soc., 79, 5238 (1957).

Preparation of N⁶ -Cyclopentyl-9-Methyl Adenine

To prepare N⁶ -cyclopenyl-9-methyl Adenine the following additional steps were taken. A mixture of 6-chloro-9-methyl Adenine (0.82 g) , cyclopentylamine (0.52 ml) , trimethylamine (0.53 ml) and ethanol. (60 ml), was refluxed for 24 hours. The solution was concentrated in vacuo to a yellow syrup. The syrup was passed through a C-18 column to give 0.78 g or 74% yield of with m.p. 108°-109° C. ¹ HNMR(Me₂ SO-d₆): δ1-2 (m, 9 H); 3.7 (S, CH₃); 7.6(d,NH); 8.1(S,1H); 8.2(S,1H).

Preparation of N⁶ -3-Pentyl-9-Methyladenine

A mixture of 6-chloro-9-methyladenine (1.5 g), 3-pentylamine (1.3 ml), trimethylamine (1.3ml) and ethanol (60 ml), was refluxed for 24 hours. The solution was concentrated and passed through a C-18 column to give a white solid having m.p. 107°-109° C.

Preparation of N⁶ -(endo-2-Norbornyl)-9-Methyladenine

A mixture of 1.5 g 6-chloro-9-methyl Adenine, 1.75 g endo-2-aminonobornane, 2.9 ml trimethylamine and 60 ml ethanol was refluxed overnight. The solution was then concentrated in vacuo and the remainder was passed through C-18 prepchromatography to give 1.6 g (75% yield) m.p. 130°-131° C. ¹ HNMR(Me₂ SO-d₆): δ1-2.6(n,10 H); 3.8(S, CH₃); 4.1(m,1H); 7.2(S,NH); 7.4(S,1H); 7.6(S,1H).

Preparation of N⁶ -(endo-2-Norbornyl)⁻ 8-Bromo-9MA

To a stirred suspension of N⁶ -(endo-2-norbornyl)⁻ 9-MA (6 g, 24.66 mmoles) 2,n 150 mL of 1M sodium acetate buffer (pH 3.9) was added a solution of bromine (3.0 ml) in 300 ml of 1M sodium acetate buffer (pH 3.9). The mixture was stirred overnight and the resulting precipitate was filtered and washed with water. To the residue was added silica gel and the suspension was evaporated to a powder. The powder was added to a silica gel column (150 g, packed with petroleum ether). The purine was eluted with 10% to 25% ethylacetate in petroleum ether. Evaporation of the appropriate fractions gave 6.7 g, 84% yield of N⁶ -(endo-2-Norbornyl)⁻ 8-Bromo-9-MA Preparation of N⁶ -(endo-2-Norbornyl)⁻ 8-Azido-9-MA

To a solution of N⁶ -(endo-2-Norbornyl)⁻ 9-Bromo-9-MA (0.72 g, 2.23 mmoles) in DMF was added sodium azide (0.91 g, 13.98 mmoles). The mixture was heated at 70°-80° C. overnight. The crude was dissolved in water, extracted with ethyl acetate, and then dried over magnesium sulfate and the organic phase was evaporated in vacuo to give 0.62 g, 98% yield.

Preparation of N⁶ -(endo-2-Norbornyl)-8-Amino-9-MA

The crude product, N⁶ -(endo-2-Norbornyl)-8-Azido-9-MA (0.5 g, 1.75 mmole) was dissolved in ethanol. The solution, in presence of 10% palladium on charcoal (1 g), was shaken with H₂ at 35 atm overnight. The suspension was filtered and evaporated to a small volume, and then poured through a C-18 column (HPLC) to give 0.36 g 80% yield of N⁶ -(endo-2-norbornyl)-8-Amino-9-MA.

Preparation of N⁶ -(endo-2-Norbornyl)-8-Oxo-9-MA

To a mixture of N⁶ (endo-2-Norbornyl)-9-Bromo-9-MA (0.15 g, 0.62 mmole) in 12 ml acetic acid was added sodium acetate (0.5 g) and 1.2 ml acetic anhydride. The mixture was allowed to reflux overnight. The mixture was then evaporated under vacuo and purified on a chromatotron using CHCl₃, stepping to 2% ethanol, and finally to 4% ethanol on 2 mm plate giving 90 mg, 75% yield of N⁶ -(endo-2-Norbornyl)-8-Oxo-9-MA.

Preparation of N⁶ -(endo-2-Norbornyl)-8-Cyclopentylamine-9-MA

To a solution of N⁶ -(endo-2-Norbornyl)-8-Bromo-9-MA (0.5 g, 1.55 mmols) in 20 ml ethanol was added 20ml of cyclopentylamine; the reaction mixture was refluxed overnight. The mixture was then evaporated under vacuo and passed through a C-18 column (HPLC) to give 0.32 g, 77% yield of N⁶ -(endo-2-Norbornyl)-8-Cyclopentylamine-9-MA.

Preparation of N⁶ -(endo-2-Norbornyl)8-Bromo-2-Chloro-9-MA

N⁶ (endo-2-Norbornyl)2-Chloropurine was first prepared as follows: A mixture of 2,6-dichloropurine (5.0 g, 26.45 mmoles) endo-2-aminobornane hydrochloride (5.0 g, 33.86 mmoles) and trimethylamine (10 ml) in absolute ethanol was refluxed for 48 hours. The solution was then cooled to room temperature and evaporated in vacuo to a white solid. The white solid was washed with water and dried to yield 6.0 g, 84% yield of N⁶ -(endo-2-Norbornyl)2-Chloropurine used as is with no further purification for next step.

A mixture of N⁶ -(endo-2-Norbornyl)-2-chloropurine (5.0 g, 18.96 mmoles), triethyl ammonium hydroxide (18.9 ml), and methyl iodide (1.41 ml, 22.68 mmoles) in dichloromethane was heated to 35° C. for 24 hours. The solution was then evaporated in vacuo and the syrup was crystallized in methanol to give 4.0 g, 76% yield of N⁶ -(endo-2-Norbornyl)2-chloro-9-MA.

To a stirred solution of N⁶ -(endo-2-Norbornyl)-2-chloro-9-MA (4.3 g, 14.4 mmoles) in acetate buffer (1 molar acetic acid and 1 M sodiums acetate mixture, 45:1 ratio respectively; pH=3.9) was added dropwise Bromine (3.12 g, 19.56 mmoles) dissolved in the acetate buffer. The reaction mixture was stirred for 72 hours; the mixture was then filtered and the solid material collected was eluted from ethyl acetate/petroleum ether on silica gel column to yield 4.9 g, 85% of N⁶ -(endo-2-Norbornyl)8-Bromo-2-Chloro-9-MA.

Preparation of N⁶ -(endo-2-Norbornyl)-8-Cyclopentyl-9-MA

To a vigorously stirred solution of 2 g (12.2 mmoles) of 4-methylamino-5-amino-6-chloropyrimidine in CHCl₃ was added dropwise over a period of 20 minutes cyclopentane carbonyl chloride (1.6 g, 12.2 mmoles). The mixture was stirred overnight and then evaporated in vacuo to a yellow syrup. The syrup was then dissolved in methanol and purified through a C-18 column (HPLC) to give 2.2 g, 71% yield of 4 methylamino-6-chloro-5-cyclopentylamido-pyrimidine.

4-methylamino-6-chloro-5-cyclopentylamido-pyrimidine 2.2 g, 8.6 mmoles) was refluxed in POCl₃ for approximately 2 hours. The solution was concentrated in vacuo to a syrup. The syrup was added dropwise twice. The aqueous mixture was then extracted with chloroform. The organic layer was evaporated and the syrup was passed through a C-18 column (HPLC) giving 1.25 g, 63% yield of 8-cyclopentyl-6-chloro-9-methyladenine.

A mixture of 8-cyclopentyl-6-chloro-9-methyladenine (0.48 g 2.0 mmoles) and endo-2-aminonorbornane hydrochloride (0.5 g, 3.4 mmoles) in absolute ethanol was refluxed for 48 hours. The mixture was then evaporated in vacuo and purified through a C-18 column (HPLC) to give 0.45 g, 71% yield of N⁶ (endo-2-Norbornyl)-8-cyclopentyl-9-MA.

Preparation of N⁶ -(endo-2-Norbornyl)-8-Chloro-9-MA

A mixture of N⁶ -(endo-2-Norbornyl)-8-bromo-9-MA (1.25 g, 3.7 mmoles) and POC13 was refluxed for 1 hour. Then the phosphorous oxychloride was removed in vacuo and the yellow solid was passed through a C-18 column (HPLC) to give 0.96 g, 84% yield of N⁶ -(endo-2-Norbornyl)-8-chloro-9-MA.

Preparation of N⁶ -(endo-2-Norbornyl)-9-[(2-hydroxyethoxy)methyl]purine.

To a solution of 6-chloropurine (6 g, 38.8 mmoles) in DMF was added sodium hydride 60% (0.93 g) over 1.5 hour period. (2-acetoxyethoxy)methyl bromide was then added at room temperature; the reaction mixture was allowed to stir for 2 hours under N₂ atmosphere. H₂ O was added and the product was extracted with ethyl acetate. The organic phase was dried over MgSO₄, filtered, and evaporated in vacuo to give a light yellow solid 7.1 g, 68% yield of 9-[(2-Acetoxy-ethoxy)methyl]-6-chloro-purine. The crude was used without further purification.

To a solution of 9-[(2-acetoxyethoxy)methyl]-6-chloropurine (5.1 g, 18.8 mmoles) in ethanol and trimethylamine was added endo-2-aminonorbornane hydrochloride (4.0 g, 27.1 mmoles). The mixture was refluxed in vacuo and the residue was purified by HPLC to give 4.70 g, 77% yield of N⁶ -(endo-2-Norbornyl)-9-[(2-acetoxyethoxy)methyl]purine.

A solution of N⁶ -(endo-2-Norbornyl)-9-[(2-acetoxyethoxy) methyl]purine (3.75 g, 10.8 mmoles) in methanol was saturated with NH₃ gas under N₂. The mixture was stirred overnight, then evaporated in vacuo to give 2.03 g, 62% yield of N⁶ -(endo-2-Norbornyl)-9-[(2-hydroxyethoxy)methyl]purine.

Preparation of 1R, 2S, 4S- endo-2-Aminonorbornane

A) As is well known in the art¹, resolution of chiral amines may be accomplished by preparing diastereomeric salts with chiral acids. The resultant diastereomeric salts show different physical properties, enabling separation by methods such as fractional crystallization or chromatography. Thus, a mixture of endo-2-aminonorbornane isomers are reacted with chiral acids (e. g., Tartaric Acid, Mandelic Acid) to form diastereomeric salts and subjected to fractional crystallization. Alternatively, chromatographic separation of these salts (e.g., silica gel) may be employed.

B) Chromatography employing chiral stationary phases (e.g., Pirkle, Beta-cyclodextrin columns) may be used to isolate the above isomers.

C) The individual isomers may be prepared individually starting from (+) and (-) endo-2-carboxylnorbornane-2,3. These carboxylic acids may be individually converted to the above isomers by the Curtius rearrangement⁴,5, wherein the carboxylic acid is replaced by an amino group. See also Berson, J., et al., J. Amer. Chem. Soc., 81, 4094-8 (1959) and J. Amer. Chem. Soc., 83, 3986-3997 (1961).

Preparation of 1R,2S,4S- and 1S,2R,4R-(N⁶ -endo-2-Norbornyl-9-methyladenine

The above prepared amines are reacted with 6-chloro-9-methyladenine as previously described.

REFERENCES

1. March, J., Advanced Organic Chemistry: Reactions, Mechanisms and Structure, Third Ed., John Wiley and Sons, 104, 1985.

2. Scheidegger, U. et al, J. Amer. Chem. Soc, 89, 894 (1967).

3. Evans, D. A. , et al. , J. Amer. Chem. Soc. , 110, 1238 (1988) .

4. Beilstein 12 (3), 160.

5. March, 984, 1985.

For purposes of this invention, designation of an optically active stereoisomer shall mean that one enantiomer is present in excess of the other enantiomer. Preferably, the mixture is greater than 90 mole percent of one enantiomer, most preferably one enantiomer is substantially pure, i.e, greater than 99 mole percent.

The invention is further illustrated by the following examples which are illustrative of various aspects of the invention. These examples are not intended as limiting the scope of the invention as defined by the appended claims.

PHARMACOLOGIC TESTING

A series of N⁶ -substituted 9-methyladenines were assayed as adenosine antagonists in A₁ and A₂ test systems (Ukena, et al, FEBS Lett. 215(2), 203-208, 1987). For activity at A₁ receptors, compounds were tested as inhibitors of the binding of N⁶ -E-[3H]-Phenylisopropyladenosine in rat brain membranes and for their ability to prevent R-PIA-induced inhibition of adenylate cyclase in rat fat cell membranes. For activity at A₂ receptors, compounds were tested as antagonists of NECA-stimulated adenylate cyclase in membranes of human platelets and rat PC12 cells.

It is known that A₁ receptors influence inhibition of adenylate cyclase in fat, brain and heart cells; whereas A₂ receptors stimulate adenylate cyclase in endothelial and smooth muscle cells. (See John W. Daly, et al, "Structure-Activity Relationship for N⁶ -Substituted Adenosines at a Brain A₁ -Adenosine Receptor With A Comparison to an A₂ -Adenosine Receptor Regulating Coronary Blood Flow," Biochemical Pharmacology, Vol. 35. No. 15, pp. 2467-2471 (1986)).

The results summarized below in Table I show that N⁶ substitution can markedly increase the potency of 9-methyladenine at adenosine receptors. The lower apparent affinity values (K_(B), K_(i)) identify the most potent compounds. The most pronounced effect is seen at A₁ receptors. For example, N⁶ -Cyclopentyl-9-methyladenine is at least 100-fold more potent than 9-methyladenine at A₁ receptors. At A₂ receptors, this compound is 5-fold more potent than 9-methyladenine in the human platelet assay. Thus, this data demonstrates the activity of a novel series of adenosine antagonists.

                                      TABLE 1                                      __________________________________________________________________________                                           A.sub.1 Effects                                                A.sub.2 Effects K.sub.B (μM)                                                                           K.sub.i (μM)                                     K.sub.B (μM) vs NECA Stimulation                                                            vs PIA INHIBITION                                                                         vs [.sup.3 H] PI                                    (Adenylate Cyclase)                                                                            (Adenylate Cyclase)                                                                       (Binding)                                           (A)     (B)     (C)        (D)                           __________________________________________________________________________       Adenine             760     570     >1000      >100                            9-Methyladenine (9-MA)                                                                             24      24      112        106                           N.sup.6 -substituted 9-methyladenines                                            N.sup.6 -cyclobutyl-0-MA                                                                           5.5     23      0.89       1.2                             N.sup.5 -Cyclopentyl-9-MA                                                                          4.9     25      1.3        0.54                            N.sup.6 -Methylcylopentyl-9-MA                                                                     45      56      9.0        2.5                             N.sup.6 -Cyclohexyl-9-MA                                                                           7.4     21      0.65       0.94                            N.sup.6 -Methyl-9-MA                                                                               150     130     220        >100                            N.sup.6 -3-Penytl-9-MA                                                                             11      53      7.6        3.3                             N.sup.6 Phenyl-9-MA 21      107     10         25                            10.                                                                              N.sup.6 -2-Fluorophenyl-9-MA                                                                       11      29      17         8.5                             N.sup.6 -2-Benzyl-9-MA                                                                             57      100     49         17                              N.sup.6 -2-Phenethyl-9-MA                                                                          170     120     >300       >100                            N.sup.6 -2-(3,4,5-Trimethoxyphenylethyl)-9-MA                                                      23      40      122        >100                            N.sup.6 -2-(3-Pyridylethyl)-9-MA                                                                   92      117     96         41                              N.sup.6 -2-(3-Thienylethyl)-9-MA                                                                   14      25      24         20                              N.sup.6 R-1-Phenyl-2-propyl-9-MA                                                                   13      25      7.2        2.5                             N.sup.6 -S-1-Phenyl-2-propyl-9-MA                                                                  23      74      23         10                            __________________________________________________________________________      (A)  Human Platelet Membranes                                                  (B)  Rat PC12 Membranes                                                        (C)  Rat Fat Cell Membranes                                                    (D)  Rat Brain Membranes                                                 

FURTHER FUNCTIONAL ASSAYS

To test the selectivity of the compounds of the invention, in vitro assays were conducted utilizing model tissues that are thought to contain homogenous populations of either the A₁ or A₂ adenosine receptors. Four examples were characterized by their ability to antagonize competitively the action of adenosine agonists in eliciting two responses: the reduction in force of contraction of guinea pig atrium (A₁); and the decrease in the contractile tone of the guinea pig taenia caecum (A₂).

The left atria from male guinea pigs were isolated, suspended between two punctate electrodes, and placed in a 20 ml organ bath that contained Krebs-Hensileit solution that was continuously gassed with 95% O₂ +5% CO₂ and maintained at 31° C. The resting tension was one gram. The atria were stimulated electrically at 1 Hz, 1 msec duration pulses at supramaximal voltage. The force of contraction was recorded isometrically.

Taenia from the guinea pig caecum were cut into lengths of 1.5-2 cm. The tissues were suspended in a 20 ml organ bath containing de Jalon's solution that was gassed with 95% O₂ +5% CO₂ and maintained at 31° C. The resting tension was 1.5 g. The contractile response was measured isotonically. Tissues were contracted with 10⁻⁷ M 5-methylfurmethide and allowed sufficient time to reach a stable contraction before addition of adenosine agonists.

The ability of the compounds to antagonize the effects of agonists was analyzed using modified Schild plots.

Although there was some sensitization of the tissue, i.e. addition of the agonist produced a larger response in the presence of high concentrations of the subject compounds, N⁶ -3-Pentyl-9-MA, N⁶ -Cyclopentyl-9-MA and N⁶ -(endo-2-Norbornyl)-9-MA did not competitively antagonize the effects of adenosine agonists in relaxing the taenia caecum. Sensitization is also observed when using high concentrations of 8-phenyltheophylline (8-PT), a non-selective adenosine receptor antagonist. 8-PT did antagonize the effects of agonists at low concentrations. The lack of competitive antagonism by the other I compounds suggests that the latter compounds do not interact appreciably with A₂ -adenosine receptors and are, thus, selective for A₁ adenosine receptors.

However, N⁶ -3-Pentyl-9-MA, N⁶ -Cyclopentyl-9-MA, N⁶ -(endo-2-Norbornyl)-9-MA and N⁶ -4-(2-thienyl)-3-butyl)-9-MA all were found to be competitive antagonists at adenosine receptors in the atria. N⁶ -3-Pentyl-9-MA and N⁶ -1-(2-thienyl)-2-butyl-9-MA also produced increases in basal force of contraction in the atria. Affinity constants (pKB) for the present compounds determined using known methods are summarized in Table 2 below:

                  TABLE 2                                                          ______________________________________                                         Drug                pK.sub.B                                                   ______________________________________                                         N.sup.6 -3-Pentyl-9-MA                                                                              5.4 ± 0.14                                             N.sup.6 -Cyclopentyl-9-MA                                                                          6.17 ± 0.11                                             N.sup.6 -(endo-2-Norbornyl)-9-MA                                                                   6.28 ± 0.09                                             N.sup.6 -1-(2-Thienyl)-2-butyl)-9-MA                                                               5.36 ± 0.1                                              ______________________________________                                    

These results show that the above examples display selectivity towards the A₁ adenosine receptor, with N⁶ -(endo-2-Norbornyl)-9-MA being the most potent antagonist.

IN VIVO ASSAY

In vitro selectivity of the present antagonists was confirmed by in vivo tests on rat heart rate and blood pressure, the former associated with A₁ receptors and the latter associated with A₂ receptors.

Rats were anesthetized with urethan and blood pressure was monitored via a carotid cannula. Drug injections were made intravenously through a jugular cannula. Blood pressure, EGC, and heart rate were recorded on a Grass polygraph.

Adenosine produced a dose dependent decrease in blood pressure and heart rate, with a concommitant increase in the P-R interval of the ECG. . Administration of N⁶ -(endo Norbornyl) -9-methyladenine attenuated the effects of subsequently administered adenosine on all parameters measured. At high doses, adenosine causes heart block; this effect was also substantially reduced by the agonist. Due to the short duration of action and direct route of administration of adenosine, it is often difficult to determine whether adenosine decreased blood pressure by causing peripheral vasodilation or by reducing cardiac output. To overcome these problems, NECA (5'-N-ethylcarboxamide adenosine), which is longer-acting and selective for A₂ adenosine receptors, was used as an adenosine receptor agonist. Prior administration of N-0861 attenuated the effects of NECA on the heart while minimally affecting the NECA-induced decrease in blood pressure. These results show that N⁶ -endo-2-Norbornyl)-9-methyladenine is a cardio-selective adenosine receptor antagonist in vivo and support the data above showing selectively of the N-6 substituted 9-methyladenines of the invention as A₁ adenosine receptor antagonists.

FURTHER RECEPTOR AFFINITY ASSAYS

Further tests to discover the affinities of test compounds at A₂ receptors were conducted. [3H]-N-ethylcarboxamido adenosine ([3H-]-NECA) was used as the radio-ligand, bovine caudate was the source of membranes, and the assay buffer was 50 mM Tris; 10 mMMgCl₂, pH 7.4.

To provide bovine caudate nuclei, bovine brains were obtained fresh from a local slaughterhouse. The caudate nuclei were dissected out and homogenized in Buffer A (50 mm Tris; 1 mm Na2-EDTA; 5 mm KCl; 1 mm MgCl₂ ; 2 mm CaCl₂ ; pH 7.4) using a Brinkman Polytron. The homogenate was centrifuged at 40,000×g for 20 minutes and washed once. The pellet was resuspended in Buffer A, incubated at 37° C. for 15 minutes, then centrifuged. The pellet was washed once more, resuspended to a protein concentration of 5-10 mg/ml in Buffer A and frozen at -70° C. until use.

The A₂ assays also contained 50 nM cyclopentyl-adenosine to block the binding of [3H]-NECA to A₁ receptors (Bruns et al, 1986) and 1 unit/ml adenosine deaminase to degrade endognous adenosines. Varying concentrations of test compounds were incubated with the appropriate radioligand and membrane source for 1 hr at room temperature.

Assays were terminated by filtration over Whatman GF/B filters that had been pre-soaked with 0.1% polyethyleneimine using a 24 port Brandell cell havester. The filters were washed three times with 3 ml of ice cold buffer and transfered to plastic scintillation vials to which 4 ml of Beckman Ready protein scintillation cocktail was added. The tubes were shaken and counted in a Beckman 3801 scintillation counter that converted cpm to dpm.

Data were analyzed by utilizing the Ligand, commercial computer program (Munson and Rodbard, 1980).

The results of these tests, expressed as the molar concentration of test compound needed to displace 50 percent of the [3HI-CHA radioligand from rat cortical A₁ receptors, are summarized in Table 3 below:

                  TABLE 3                                                          ______________________________________                                         Adenosine Antagonists                                                                                      Rat Cortical                                       Sample                      Binding Constant                                   No.   Name                  Ki (M)                                             ______________________________________                                         0861  N.sup.6 -(endo-2-norbornyl)-9-MA                                                                     11.6 × 10.sup.-8                             0913  N.sup.6 -(endo-2-norbornyl)-2-chloro-9-MA                                                            10.5 × 10.sup.-8                             0966  N.sup.6 -2,2-diphenylethyl-9-MA                                                                      >10.sup.-5                                         0967  N.sup.6 -2(2-chlorophenylethyl)9-MA                                                                  >10.sup.-5                                         0982  N.sup.6 -2-Aminoethyl-9-MA                                                                           >10.sup.-5                                         0983  N.sup.6 -(2,2-N-dimethylethyl)-9-MA                                                                  >10.sup.-5                                         0840  N.sup.6 -cyclopentyl-9-MA                                                                            37.5 × 10.sup.-8                             0984  N.sup.6 -R-1-phenyl-1-ethyl-9-MA                                                                     >10.sup.-5                                         0985  N.sup.6 -S-1-phenyl-1-ethyl-9-MA                                                                     >10.sup.-4                                         0986  N.sup.6 -S-1-phenyl-2-propyl-9-MA                                                                    >10.sup.-5                                         0987  N6 2-thienyl-9-MA     >10.sup.-4                                         0988  N6(4-chloro-2-methylphenyl)-9-MA                                                                     >10.sup.-5                                         0989  N.sup.6 -2-(3-ethylindole)-9-MA                                                                      >10.sup.-5                                         0990  N.sup.6 -2-(phenethyl)9-MA                                                                           >10.sup.-5                                         1001  N.sup.6 -(endo-2-norbornyl)-8-oxo-9-MA                                                               ≈10.sup.-5                                 1002  N.sup.6 -2-(3,4,5-trimethoxyphenyl)ethyl-9-                                                          >10.sup.-5                                               MA                                                                       1003  N.sup.6 -(endo-2-norbornyl)-8-bromo-9-MA                                                              1.3 × 10.sup.-8                             1004  N.sup.6 -1-carboxy-1-butyl-9-MA                                                                      >10.sup.-4                                         1005  N.sup.6 -(endo-2-norbornyl)-8-amino-9-MA                                                               87 × 10.sup.-8                             1006  N.sup.6 -(endo-2-norbornyl)-8-carboxy-9-                                                             >10.sup.-5                                               MA Sodium Salt                                                           1059  N.sup.6 -(endo-2-norbornyl)9-[(2 hydroxy-                                                              49 × 10.sup.-8                                   ethoxy) methyl]adenine                                                   1060  N.sup.6 -(endo-2-norbornyl)-8-thio-9-MA                                                                37 × 10.sup.-8                             1061  N.sup.6 -(endo-2-norbornyl)-8-chloro-9-MA                                                             1.5 × 10.sup.-8                             1062  N.sup.6 -(endo-2-norbornyl)-8-sulfonate-9-                                                           >10.sup.-4                                               MA Sodium Salt                                                           1063  N.sup.6 -(Endo-2-norbornyl)-2-oxo-9-MA                                                                112 × 10.sup.-8                             1064  N.sup.6 -(endo-2-norbornyl)-8-cyclopentyl-                                                            190 × 10.sup.-8                                   amine-9-MA                                                               0964  N.sup.6 -(endo-2-norbornyl)-8-cyclopentyl-                                                             24 × 10.sup.-8                                   9-MA                                                                     0965  N.sup.6 -cyclopentyl-8-cyclopentyl-9-MA                                                              14.1 × 10.sup.-8                             0978  N.sup.6 -(exo-2-norbornyl)-9-MA                                                                        43 × 10.sup.-8                             ______________________________________                                    

The compounds in Table 3 for which a solution having a concentration greater than 10⁻⁵ M was required to displace 50 percent of the radioligand are deemed ineffective as A₁ adenosine receptor antagonists.

In further experiments designed to determine the selectivity of N⁶ -endo-2-Norbornyl-9-methyl adenine at A₁ receptors, [3HI -cyclohexyladenosine ( [3H]-CHA) was used as the radioligand, rat cortical membranes were the receptor source, and the assay buffer was 50 mM Tris; 2 mM MgCl₂ pH 7.4.

Male Sprague Dawley rats were killed by decapitation and the brains removed. The cerebral cortices were homogenized in 50 mm Tris; 2mm MgCl₂ (pH 7.4), and centrifuged at 40,000×g for 10 minutes. The pellet was washed once, resuspended in Tris/MgCl₂ and incubated with 8 units/ml adenosine deaminase at 37° C. for 30 minutes. The homogenate was centrifuged, washed once, resuspended to a protein concentration of 5-10 mg/ml and frozen at -70° C. until use. The results in Table 4 below show that the test compound has 170 times more affinity for A₁ receptors than for A₂ receptors.

                  TABLE 4                                                          ______________________________________                                         Selectivity of N.sup.6 -endo-2-Norbornyl-9-MA                                  Bovine Caudate Binding                                                         At A.sub.1 Receptors                                                                        At A.sub.2 Receptors                                              K.sub.i (M)  K.sub.i (M)                                                       ______________________________________                                         4.1 × 10.sup.-8 M                                                                     6.96 × 10.sup.-6 M                                                     A1/A2 =    5.89 - 10.sup.-3                                                    =          170 fold selective                                                             for A.sub.1 receptors                                    ______________________________________                                    

REFERENCES

Munson, Peter J. and Rodbard, David (1980). "Ligand: A Versatile Computerized Approach for Characterizing Ligand binding Systems." Anal. Biochem. 107:220-239.

Bruns, Robert F., Lee, Gina H., and Pugsley, Thomas A. (1986) "Characterization of the A₂ Adenosine Receptor Labeled by ³ H-NeCA in Rat Striatal Membranes," Mol. Pharmacol. 29:331-346.

These N⁶ -substituted adenines are antagonists of A₂ adenosine receptor-mediated stimulation of adenylate cyclase in A₂ -adenosine receptors and antagonists of A₁ -adenosine receptor-mediated inhibition of adenylate cyclase. These compounds are useful in reversal of adenosine-mediated lipolysis, reversal of adenosine-mediated deleterious cardiovascular effects (conduction defects, hypotension), reversal of adenosine-mediated vascular actions in kidney, bronchodilation, antiarrhythmic action, reversal of adenomediated relaxation of smooth muscle, anti-narcoleptic action, CNS stimulation, and blockade of adenosine mediated inhibition of neurotransmitter release.

While not wishing to be bound by theory as to the mechanism of action involved, it has been found that the optically active compounds covered by this invention such as the 1R,2S,4S- and 1S,2R,4R-(endo-2-Norbornyl)-9-methyladenines, especially those substituted with hydrogen and halogen at the 8 position, show greatly increased selectivity as A₁ adenosine receptor antagonists over racemic mixtures of the compound. Of the two groups of enantiomers, the 1R,2S,4S Norbornyl compounds show the greatest increase in selectivity for A₁ adenosine receptors. The compounds of this invention have been found to be useful in the treatment of glaucoma.

While particular embodiments of the invention have been described it will be understood of course that the invention is not limited thereto since many obvious modifications can be made and it is intended to include within this invention any such modifications as will fall within the scope of the appended claims. 

What is claimed is:
 1. A compound having the formula: ##STR6## or stereoisomers thereof; wherein X is hydrogen.
 2. A compound of claim 1 wherein said compound is one of:N⁶ -[endo(1R,2S,4S)-2-norbornyl]-9-methyladenine or N⁶ -[endo(1S,2R,4R)-2-norbornyl]-9-methyladenine. 